Project Summary/Abstract Development of the adaptive immune response requires generation of a T cell repertoire capable of recognizing a wide range of potential pathogens. However, this anticipatory repertoire has to be generated without ever seeing these pathogens. This is achieved by the process of positive selection of T cells in the thymus, which ensures the ability of a TCR to recognize peptide antigens presented by self-MHC molecules. Positive selection, which takes several days to complete, requires a sustained signal through Ca2+ and Erk and involves a weak interaction between a TCR on a thymocyte and a self-peptide/MHC complex on the surface of a cortical epithelial cell. We have recently identified a naturally occurring positive selecting self-peptide for AND CD4+ T cells, gp250. The solution 3D KD of the gp250/I-Ek:AND interaction was shown to be extremely weak. Despite its apparent weak affinity, gp250/I-Ek was able to induce a sustained Ca2+ signal and in vitro positive selection of AND thymocytes, whereas, the MCC agonist induced a transient signal, and negative selection. It is not known how a weak TCR:pMHC interaction can transduce sustained signaling in thymocytes. Essentially nothing is known about the molecular details for a TCR:positive selecting pMHC ligand interaction. In Aim I, we propose studies to determine biophysically and structurally how the AND TCR binds to the positive selecting gp250/I-Ek ligand in comparison to the agonist ligand, MCC/I-Ek. We will determine the 2D affinity of these ligands as well as identify the threshold of affinity between positive and negative selection. The structural details of how the AND TCR binds gp250/I-Ek and MCC/I-Ek will be studied using a series of I-Ek point mutants, and the solving the crystal structures of the AND:gp250/I-Ek and AND:MCC/I-Ek co-complexes. The studies in Aim II investigate the novel observation that a voltage-gated sodium channel (composed of the SCN5a pore subunit and SCN4b regulatory subunit) induces a sustained Ca2+ influx and positive selection of AND T cells. Specific inhibition of the voltage gated sodium channel inhibited a gp250 stimulated sustained Ca2+ influx signal, and importantly blocked positive selection of AND thymocytes in reaggregate cultures. shRNA knockdown of SCN5a inhibited CD4 T cell positive selection in vivo. We hypothesize that a voltage gated sodium channel is critically involved in positive selection by activating a voltage gated Ca2+ channel in thymocytes, resulting in weak ligands being able to induce a sustained Ca2+ signal. The mechanism by which SCN5a/SCN4b are affecting signaling in thymocytes during positive selection will be determined electrophysiologically and biologically, and will involve the generation of conditional knockout mice for both genes. These studies will provide key new insights into how the process of positive selection occurs at a structural and signaling level, with the possibility of yielding molecular targets for the manipulation of unwanted T cells, including T cells involved in autoimmunity.